Detection of SNPs by PCR using fluorescently labeled melting probes has been previously described (see, e.g., Huang et al., 2011, PLoS ONE 6(4) e19206 and Luo et al., 2011, J. Clin. Microbiol., 49(9)3132-3138). Traditional melting works by analyzing the post-PCR melting temperature of a fluorescently labeled probe with target in the absence of a SNP and in the presence of a SNP. A well-designed melting probe yields a melting temperature (Tm) which is highest in the absence of a SNP, and any base pair changes that occur in the probe binding region causes a shift (decrease) in melting temperature. The shift in melting temperature can be used to distinguish of wild type (WT) and mutant (MT) targets.
Unfortunately, some SNPs of interest are located in non-conserved gene regions where near-by sequence heterogeneity causes an unwanted shift in probe melting temperature. This makes it difficult to distinguish WT sequences with silent mutations from the relevant SNPs of interest. SNPs associated with micro-organism drug resistance are some examples where specific SNPs conferring drug resistance are located in gene regions that contain near-by WT silent mutations. In such a case, any silent mutation(s) in the probe binding region near the SNP of interest could also generate a shift in probe melting temperature and yield a false-positive result. Thus, there is a need for more accurate and effective way to detect only the SNP of interest in a target nucleic acid. Embodiments of the present invention can solve the existing problems of sequence heterogeneity and enable a well-designed melting probe to detect only the SNP of interest without interference from nearby silent mutations.